PROJECT SUMMARY/ABSTRACT Clostridium difficile is the main cause of antibiotic-associated diarrhea. Since 2003, the incidence and severity of C. difficile infection (CDI) has risen in the U.S. and globally. In the U.S. in 2011, there were ~29,000 deaths from ~500,000 CDI cases. In 2013 the CDC designated C. difficile as an Urgent Threat. These trends were related to the emergence of epidemic strains, in particular epidemic 027. Epidemic 027 causes about a third of CDI in the U.S. and has a tendency to cause severe disease. The firstline drug metronidazole is now more prone to fail today than 30 years ago. Our study suggests that metronidazole resistance is one hitherto overlooked factor that is driving treatment failures. This proposal seeks to understand the impact of resistance on treatment responses to metronidazole and the genetic basis for metronidazole resistance (MTZ-R) in C. difficile. We discovered that a subset of epidemic 027 strains display decreased susceptibility to metronidazole, when tested in fresh heme. Using fresh heme, we identified other metronidazole-resistant epidemic strains from different lineages. Heme induces the expression of metronidazole resistance that is otherwise not detected. We demonstrate that resistance occurs during therapy in animals and it could be selected in lab- media. This work has significant implications for public health care, because the impact of metronidazole resistance is not well characterized. Our study will provide insights to how resistance affects treatment responses to metronidazole and will unveil how it evolved in epidemic 027 and other lineages. Aim 1. To characterize the clinical impact of MTZ-R. This uses a biobank of patient stools to assess colonization with resistant strains, the on-therapy rise of resistance and if it hinders responses to MTZ. Further validations are done in a clinically reflective in vitro and animal models of CDI. Aim 2. To test if the low colonic levels of MTZ facilitates on-therapy development of MTZ-R and if it is ineffective in CDI with MTZ-R strains. MTZ will be compared to a non-absorbed MTZ analog to quantify the emergence of MTZ-R in low and high drug levels in CDI animals. The non-absorbed MTZ analog will also test the importance of high drug levels in the colon for resolving CDI due to resistant strains. Aim 3. To elucidate the evolution of MTZ-R from laboratory and patient isolates. This aim moves the C. difficile field forward to link phenotypes to their resistance genotypes and elucidates the evolutionary basis of metronidazole resistance in three settings: lab-media, animal models and clinical isolates from patients. It then applies computational genomics and molecular experimentation to identify and validate convergent or divergent paths to resistance. Public health. The